Intramural delivery of nitric oxide enhancer for inhibiting lesion formation after vascular injury

ABSTRACT

Vessels suffering vascular injury from angioplasty are treated with L-arginine intramurally. The incidents associated with restenosis are substantially reduced providing for a reduced incidence of restenosis as a result of the injury.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/695,792, filed Aug. 12, 1996, which is a continuation-in-part of08/556,035, filed Nov. 9, 1995, which is a continuation-in-part ofapplication Ser. No. 08/336,159, filed Nov. 8, 1994, now abandoned,which is a continuation-in-part of application Ser. No. 076,312, filedJun. 11, 1993, now U.S. Pat. No. 5,428,070.

BACKGROUND

The long term benefit of coronary balloon angioplasty and atherectomy islimited by the considerably high occurrence of symptomatic restenosis(40-50%) 3 to 6 months following the procedure (Holmes et al. Am. J.Cardiol. (1984) 53:77C-81C). Restenosis is in part due to myointimalhyperplasia, a process that narrows the vessel lumen and which ischaracterized by vascular smooth muscle cell migration and proliferation(Forrester et al. J. Am. Coll. Cardiiol. (1991) 17:758-769). Medicaltherapies to prevent restenosis have been uniformly unsuccessful.Intravascular stents have been successfully used to achieve optimallumen gain, and to prevent significant remodeling. However, intimalthickening still plays a significant role in stent restenosis.

The vascular architecture is maintained or remodeled in response to thechanges in the balance of paracrine factors. One of the substances thatparticipates in vascular homeostasis is endothelium derived nitric oxide(NO). NO is synthesized from the amino acid L-arginine by NO synthase.NO relaxes vascular smooth muscle and inhibits its proliferation. Inaddition, NO inhibits the interaction of circulating blood elements withthe vessel wall. NO activity is reduced in hypercholesterolemia andafter vascular injury. We have shown that administration of the NOprecursor (L-arginine) has been shown to restore vascular NO activity inanimals and in humans with endothelial vasodilator dysfunction due tohypercholesterolemia, atherosclerosis, or restenosis. Chronicenhancement of NO activity (by oral administration of L-arginine) isassociated with a significant reduction in intimal thickening due tohypercholesterolemia and/or vascular injury. The observations associatedwith the oral administration are limited to systemic action. Cooke, etal, J Clin Invest 1992; 90:1168-72; McNamara, et al, Biochem Biophys ResComm 1993; 193:291-6; Taguchi, et al, Life Sciences 1993; 53:PL387-92;Tarry and Makhoul, et al, Arterioscler Thromb 1994; 14:938-43; Hamon, etal, Circulation 1994; 90:1357-62; Wang, et al, J Am Coll Cardiol 1994;23:452-8.

However, oral administration of L-arginine has potential systemicside-effects. These side-effects include increases in growth hormone andinsulin release--this could, potentially, exacerbate hyperglycemia inpatients with diabetes (which is a large segment of the patientpopulation that requires balloon angioplasty for coronary or peripheralartery disease). In addition, there is evidence that in high doses (30grams daily) oral L-arginine can increase the proliferation of tumorcells in human breast cancer. Accordingly, it would be beneficial todevelop an approach which would allow one to enhance NO activityselectively in the vessel wall where this effect is needed withouthaving systemic side-effects. We have developed an approach to diminishthe incidence of restenosis resulting from angioplasty and atherectomy,using arginine to enhance NO activity in the vessel wall, while at thesame time avoiding potential systemic side-effects.

BRIEF DESCRIPTION OF THE RELEVANT LITERATURE

Intravenous oral administration of L-arginine can enhance the release ofendothelium-derived nitric oxide from vessels of animals or humans withhypercholesterolemia and/or atherosclerosis (1-8). Chronic oraladministration of L-arginine also inhibits the development ofatherosclerosis in hypercholesterolemic animals (9-10). Oraladministration of L-arginine also inhibits restenosis following ballooninjury (11-13) as does oral L-arginine combined with application ofL-arginine to the external surface of the vessel using a pluronic gel(14). However, oral or intravenous administration of L-arginine hassystemic side-effects. Oral or intravenous L-arginine is known to inducethe release of growth hormone and insulin; this could potentiallyexacerbate hyperglycemia in patients with diabetes. Moreover, one studyhas indicated that high doses of oral arginine can increase theproliferation of tumor cells in human breast cancer.

We have shown that intravascular administration of a plasmid constructcontaining the gene encoding nitric oxide synthase can increase NOproduction locally in the vessel wall and will inhibit restenosis in therat carotid (15). However, this approach required direct exposure of thevessel and surgical arteriotomy as well as prolonged installation of thesolution containing the NOS gene. This approach would be impractical forpreventing restenosis in coronary arteries. Others have shown thatadministration of drugs consisting of nitric oxide, or releasing nitricoxide, can inhibit restenosis after angioplasty. Chronic inhalation ofnitric oxide inhibits restenosis following balloon-induced vascularinjury of the rat carotid artery (16). Oral administration of NO donors(drugs which release nitric oxide) inhibits restenosis in rat and pigmodels of balloon angioplasty-induced vascular injury (17,18). However,oral or inhaled administration of nitric oxide or nitric oxide donorshave systemic effects (hypotension, headache); and are susceptible todrug tolerance (lack of effect of drug after prolonged administration.There is even some evidence in animal models that NO donors mayaccelerate atherosclerosis, possibly by suppressing endogenous NOactivity in the vessel wall (19,20).

1. Girerd X J, Hirsch A T, Cooke J P, Dzau V J, Creager M A. L-arginineaugments endothelium-dependent vasodilation in cholesterol-fed rabbits.Circ Res 1990;67:1301-1308.

2. Cooke J P, Andon N A, Girerd X J, Hirsch A T, Creager M A: Argininerestores cholinergic relaxation of hypercholesterolemic rabbit thoracicaorta. Circulation 1991;83:1057-62.

3. Rossitch E, Jr., Alexander E., III, Black P, Cooke J P. L-argininenormalizes endothelial function in cerebral vessels fromhypercholesterolemic rabbits. J Clin Invest 1991;87:1295-1299.

4. Drexler H, Zeiher A M, Meinzer K, Just H. Correction of endothelialdysfunction in coronary microcirculation of hypercholesterolemicpatients by L-arginine. Lancet 1991;338:1546-1550.

5. Creager M A Gallagher S J, Girerd X J, et al. L-arginine improvesendothelium-dependent vasodilation in hypercholesterolemic humans. JClin Invest 1992;90:1248-53.

6. Kuo L, Davis M J, Cannon M S, Chilian W M. Pathophysiologicalconsequences of atherosclerosis extend into the coronarymicrocirculation. Restoration of endothelium-dependent responses byL-arginine. Circ Res 1992;70(3):465-76.

7. Tsao P S, McEvoy L M, Drexler H, Butcher E C, Cooke J P. Enhancedendothelial adhesiveness in hypercholesterolemia is attenuated byL-arginine. Circulation 1994;89:2176-82.

8. Drexler H, Fischell T A, Pinto F J, Chenzbraun A, Botas J, Cooke J P,Alderman E L. Effect of L-arginine on coronary endothelial function incardiac transplant recipients: Relation to vessel wall morphology.Circulation 1994;89:1615-1623.

9. Cooke J P, Singer A H, Tsao P S, Zera P, Rowan R A, Billingham M E.Anti-atherogenic effects of L-arginine in the hypercholesterolemicrabbit. J Clin Invest 1992;90:1168-72.

10. Wang B, Singer A, Tsao P, Drexler H, Kosek J, Cooke J P: Dietaryarginine prevents

atherogenesis in the coronary artery of the hypercholesterolemic rabbit.J Am Coll Cardiol 1994;23:452-58.

11. McNamara D B, Bedi B, Aurora H, Tena L, Ignarro L J, Kadowitz P J,Akers D L. L-arginine inhibits balloon catheter-induced intimalhyperplasia. Biochem Biophys Res Comm 1993;193(1):291-6.

12. Tarry W C, Makhoul R G. L-arginine improves endothelium-dependentvasorelaxation and reduces intimal hyperplasia after balloonangioplasty. Arterioscler Thromb 1994;14(6):938-43.

13. Hamon M, Vallet B, Bauters Ch, Wernert N, McFadden E F, Lablanche JM, Dupuis D, Bertrand M E. Long-term oral administration of L-argininereduces intimal thickening and enhances neoendothelium-dependentacetylcholine-induced relaxation after arterial injury. Circulation1994;90:1357-62.

14. Taguchi J, Abe J, Okazaki H, Takuwa Y, Kurokawa K. L-arginineinhibits neointimal formation following balloon injury.1993;53(23):PL387-92.

15. von der Leyen H, Gibbons G H, Morishita R, Lewis N P, Zhang L,Nakajima M, Kaneda Y, Cooke J P, Dzau V J: Gene therapy inhibitingneointimal vascular lesion: In vivo transfer of endothelial cell nitricoxide synthase gene. Proc Natl Acad Sci USA 1995;92:1137-41.

16. Lee J S, Adrie C, Jacob H J, Roberts J D, Jr., Zapol W M, Bloch K D.Chronic inhalation of nitric oxide inhibits neointimal formation afterballoon-induced arterial injury. Circ Res 1996;78(2):337-42.

17. Seki J, Nishio M, Kato Y, Motoyama Y, Yoshida K. FK409, a new nitricoxide donor, suppresses smooth muscle proliferation in the rat model ofballoon angioplasty. Arterosclerosis 1995;117(1):97-106.

18. Groves P H, Banning A P, Penny W J, Newby A C, Cheadle H A, Lewis MJ. The effects of exogenous nitric oxide on smooth muscle cellproliferation following porcine carotid angioplasty. Cardio Res1995:30(1):87-96.

19. Munzel t, Syegh H, Freeman B A, Tarpey M M, Harrison D G. Evidencefor enhanced vascular superoxide anion production in nitrate tolerance.A novel mechanism underlying tolerance and cross-tolerance. J. ofClinical Investigation, 1995; (1):187-94

20. Bult H, Buyssens N, DeMeyer G R Y, Jordaens F H, Herman A G. Effectsof chronic treatment with a source of exogenous nitric oxide on therelease of endothelium-derived relaxing factor by aortae from normal andhypercholesterolemic rabbits. Elsevier Science Publishers B. V.(Biiomedical Division) Nitric oxide from L-arginine: a bioregulatorysystem. Moncada S, Higgs, editors. 1990; Chapter 13, pp. 101-106

SUMMARY OF THE INVENTION

Methods and devices are provided for inhibiting the pathology associatedwith vascular injury, particularly during angioplasty and atherectomy.An NO precursor, particularly L-arginine, is intramurally introducedinto the walls of the injured vessel in proximity to the injury in anamount to inhibit the pathology, e.g. restenosis, associated with thevascular injury. Various conventional delivery devices may be used forintramural delivery of the NO precursor, which are loaded with the NOprecursor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic diagrams explaining the treatment andexperimental protocol of the acute study (FIG. 1A) and the chronic study(FIG. 1B).

FIG. 2A shows the endothelium-dependent vasomotion before and followingthe local delivery of L-arginine. The Y axis shows % constriction anddilatation, the X axis the course of the experiment. Before theL-arginine there was only a minor change in diameter in both iliacarteries. Following the local delivery of L-arginine, treated segmentsshowed significant dilatation. The control segments are constricted.*=p<0.001. FIG. 2B is a graph showing that the vessel segments distal tothe delivery site did show only minor changes in vessel diameter toacetylcholine and were not affected by the L-arginine delivery.

FIG. 3 is a representative aortogram in the hypercholesterolemic rabbit.Before Ach infusion (left panel, 10⁻⁵ M), the vessel diameter of theiliac arteries are identical. Following the local drug delivery (rightpanel), the right iliac artery dilates to the same dose of Ach afterreceiving L-arginine.

FIG. 4 is a graph showing maximum nitric oxide production 1 hr and 1week following drug delivery. Vessels treated with L-arginine (stripedbars) showed significantly higher production of nitric oxide compared tovessels treated with vehicle (dark bars) (*p<0.04, **p<0/01). Absolutevalues 1 week after the delivery were considerably higher compared tothose obtained 1 hr after the delivery of L-arginine.

FIG. 5 is a bar graph of Intima/Media ratios at 2 and 4 weeks followinglocal drug delivery. Intima/Media ratios were significantly lower in theL-arginine treated groups after 2 and 4 weeks in comparison tovehicle-treated vessels (*p<0.04, **p<0.01).

FIG. 6 depicts low power microphotographs of iliac arteries ofhypercholesterolemic rabbits 4 weeks after balloon catheter injury andlocal drug delivery. Intimal thickening is markedly reduced in thevessel segment treated with L-arginine (left) in a comparison to thattreated with vehicle (right).

FIG. 7 are histograms illustrating the percentage of intimal lesionoccupied by macrophages. In vessel segments treated with L-arginine(striped bars) macrophage accumulation did not exceed 20% of the intimalarea. By contrast, in vehicle treated segments, macrophages occupied upto 70% of the intimal area in some cases.

FIG. 8 is a fragmentary view, partially in section, of a drug deliveryapparatus for use in the subject invention positioned in a blood vesselwith the dilatation balloon in its inflated state and containing asolution of an NO precursor.

FIG. 9 is a fragmentary view, partially in section, of the NO precursordrug delivery apparatus positioned in a blood vessel and embodyingiontophoresis means to transport the drug across the balloon surface.

FIG. 10 is a perspective view of a catheter loaded with a stent in acoronary artery narrowed by a lesion.

FIG. 11 is a perspective view of an expanded stent holding the lumen ofthe coronary artery open.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and devices are provided for the treatment of pathologiesassociated with vascular injury, particularly in relation to angioplastyand atherectomy. Of particular interest is the injury referred to asrestenosis, which results from the migration and proliferation ofvascular smooth muscle cells into the intima of the vessel as well asaccretions associated with the atherosclerosis.

The method provides introducing into the vessel walls at the site ofinjury an NO precursor, which results in the enhancement of NOproduction in the cells at the site of injury. Various delivery systemsmay be employed which result in the NO forming agent infusing into thevessel wall, and being available to the cells for NO production. Deviceswhich may be employed include drug delivery balloons, e.g. porous,sonophoretic, and iontophoretic balloons, as exemplified by the devicesdepicted in WO92/11895, WO95/05866 and WO96/08286, as well as suchcommercial devices as DISPATCH® (Scimed). See also Santoian et al.,Cath. Cardiov. Diag. (1993) 30:348-354; Muller et al., J. Am. Coll.Cardiol. (1992) 20:460-466; and Ortiz et al., Circulation (1994)89:1518-1522. Also, stents may be employed where the stent carries theNO precursor agent. The stent is conveniently introduced with acatheter, so that both short and long term delivery of the NO precursoragent can be provided for enhanced protection against blockage.

The use of catheters for the delivery of the NO precursor agent will beconsidered first. The NO precursor agent is introduced in a deliveryballoon for transport by a catheter to the site of injury. The balloonmay then be expanded under pressure driving the agent from the ballooninto the surrounding vessel wall. The amount of agent which is employedmay vary depending upon the nature of the agent, the region to betreated, and the loss of the agent from the region. The infusion of theagent is maintained for sufficient time to ensure that the cells andextracellular matrix in the injury region are exposed to the agent, soas to enhance the production of NO by these cells.

The agent may have a single active ingredient or be a combination ofactive ingredients. Of particular interest are the amino acids,L-arginine and L-lysine, individually or in combination, as a mixture oras an oligopeptide, or a biologically equivalent compound, such as lowmolecular weight oligopeptides, having from about 2-10, usually 2-6amino acids, or acetylated amino acids and oligopeptides, etc.

Other agents may be employed to enhance the amount of nitric oxide,either directly by enhancing production of nitric oxide, e.g. byenhancing absorption, and/or to enhance the activity of NO synthase, orto protect NO from degradation. Generally, the dose range will be about0.2 to 0.05 the amount that would be administered orally on a dailydosage. These compounds include vitamin B₆ (5-25 mg), folate(0.04-1 mg),vitamin B₁₂ (0.05-0.1 mg), cysteine or N-acetyl cysteine (20-100 mg),vitamin C (25-200 mg), coenzyme Q (2.5-9 mg), glutathione (5-25 mg),vitamin E (20-100 I.U.), β-carotene (1-2500 I.U.) or otherphysiologically acceptable antioxidants, such as tocopherols, phenoliccompounds, thiols, and ubiquinones. These additional additives willgenerally be present in relatively small amount in the formulation,generally being present in less than about 10 weight percent, moreusually less than about 5 weight percent, frequently less than about 1percent, and in at least about 0.01 weight percent of the nitric oxideprecursor.

A physiologically acceptable medium will be employed, normally anaqueous medium, which may be deionized water, saline, phosphate bufferedsaline, etc. The amount of the active NO precursor agent will varydepending upon the particular agent employed, the other additivespresent, etc. Generally, as exemplified by L-arginine, at least about 50mg will be present, and not more than about 5 g, usually at least about100 mg, and not more than about 2 g, frequently at least about 500 mg.The concentration may be varied widely, generally ranging from about20-500, more usually from about 50-250 g/l.

The time for the treatment will usually be at least about 2 minutes, andnot more than about 0.5 hour, generally ranging from about 5-15 minutes.The rate of introduction will generally range from about 0.05-5 ml/min,depending upon all of the other factors.

The subject methodology is employed with hosts who have sufferedvascular injury, as caused by angioplasty and atherectomies. The timefor the administration of the NO precursor agent may be varied widely,providing a single administration or multiple administrations over arelatively short time period in relation to the time of injury.Generally, treatment may be before, concurrently or after the injury,usually within 2 weeks of the injury, if before, and not more than about8 weeks, usually not more than about 6 weeks, preferably in the range of0-6 weeks (where 0 intends concurrently or shortly after the priorprocedure, within 6 hours).

For the most part, the patients will be suffering from variousconditions associated with narrowed vessels, particularlyhypercholesterolemia, diabetes, tobacco use and hypertension. Thus, onewill normally be dealing with vessels which are narrowed to varyingdegrees as a result of the accumulation of plaque at the vessel wall.

It is found that with one treatment of the NO precursor agent at orabout the time of the injury, before or shortly thereafter, one canobserve enhanced vascular NO production and reduced intimal thickening,so as to substantially reduce the potential for restenosis.

In conjunction with the intramural deliver of the NO precursor agent bythe catheter, a stent may be introduced at the site of vascular injury.The stent may be biodegradable or non-biodegradable, may be preparedfrom various materials, such as metals, ceramics, plastics orcombinations thereof. Biodegradable plastics, such as polyesters ofhydroxycarboxylic acids, are of particular interest. Numerous stentshave been reported in the literature and have found commercialacceptance.

Depending on the nature of the stent, the stent may have the NOprecursor agent incorporated in the body of the stent or coated thereon.For incorporation, normally a biodegradable plastic stent will be usedwhich will release the NO precursor agent while supporting the vesseland protecting against restenosis. In the fabrication of the stent, thebiodegradable matrix may be formed by any convenient means known in theart. Alternatively, the stent may be coated with the NO precursor agent,using an adhesive or coating which will allow for controlled release ofthe NO precursor agent. The stent may be dipped, sprayed or otherwisecoated with a composition containing the NO precursor agent and amatrix, such as the biodegradable polymers described above, aphysiologically acceptable adhesive, proteins, polysaccharides or thelike. By appropriate choice of the material for the stent and/or thecoating comprising the NO precursor agent, a physiologically activeamount of the NO precursor agent may be maintained at the site of thevascular injury, usually at least one day and up to a week or more.

The amount of the NO precursor agent will be determined empirically inaccordance with known techniques using animal models. In view of thevarious compositions which may be used as the NO precursor agent,adhesive, matrix, and the like, no specific concentration of NOprecursor agent on the stent can be stated. However, the amount of theNO precursor agent employed should provide a physiologically effectiveamount to reduce proliferation of vascular smooth muscle cells andmaintain the dilation of the vessel, while preventing restenosis.

As indicated, various delivery devices may be employed for the deliveryof the active agent. FIG. 8 illustrates the drug delivery apparatus withthe balloon 12 in its inflated state and within an arterial vessel inwhich the vessel walls are indicated by the reference numeral 15. Duringpercutaneous transluminal coronary angioplasty ("PCTA") procedures, theguide wire 10 is first inserted into the selected artery to a point pastthe stenotic lesion. The dilatation catheter including the catheter body11 and the balloon 12 is then advanced along the guide wire 10 to thedesired position in the arterial system in which the balloon portion 12traverses or crosses the stenotic lesion. The balloon 12 is theninflated by introducing the NO precursor solution through the balloonlumen 14 into the interior chamber 13 of the balloon 12. Duringinflation, the outer surfaces of the balloon 12 press outwardly againstthe inner surfaces of the vessel wall 15 to expand or dilate the vesselin the area of the stenotic lesion, thus performing the angioplastyportion of the method as well as the intramural introduction of the NOprecursor into the vessel wall.

The porous balloon may be made from any of the conventional materialsused for this purpose. These include cellulose acetate, polyvinylchloride, polysulfone, polyacrylonitrile, polyurethanes, natural andsynthetic elastomers, polyolefins, polyesters, fluoropolymers, etc.Usually the film thickness will be in the range of about 10 Å to 1μ,with a nominal pore size of about 0.05 to 1μ. Alternatively, a localdrug delivery system may be employed where the agent is delivered to thevessel wall by channels that are on the exterior surface of the balloon.The balloon is placed into the diseased vessel segment as describedabove. The balloon is then inflated in the usual manner (using saline,usually containing a contrast agent), placing the channels (on thesurface of the balloon) in contact with the vessel wall. The NOprecursor solution is then infused under pressure into the channels.Perforations in the channels allow the solution to exit and jet into thevessel wall under pressure to enhance intramural delivery.

Alternatively, a local drug delivery system may be employed where theagent is delivered to the vessel wall by channels that are on theexterior surface of the balloon. The balloon is placed into the diseasedvessel segment as described above. The balloon is then inflated in theusual manner (using saline, usually containing a contrast agent),placing the channels (on the surface of the balloon) in contact with thevessel wall. The NO precursor solution is then infused under pressureinto the channels. Perforations in the channels allow the solution toexit and jet into the vessel wall under pressure to enhance intramuraldelivery.

Alternatively, an iontophoretic approach may be used. FIG. 9 illustratesa structure utilizing iontophoresis to assist in driving the active NOprecursor across the balloon wall 26 and into contact with the vesselwalls 15. One electrode 28, the catheter electrode, is located on orwithin the catheter body 11, while the other electrode 31, the bodysurface electrode, is located on the body surface or within the body ofthe patient. An electrical current for the iontophoretic process isproduced between the electrodes 28 and 31 by an external power source 30through the electrical leads 29 and 33, respectively. Direct current maybe used, although other wave forms are also utilized (e.g., a series ofrectangular waves producing a frequency of 100 Hz or greater).

During operation of the iontophoretic device, the balloon 26 is firstpositioned across the stenotic lesion. The balloon interior 27 is theninflated with the drug in the lumen 23. As the balloon expands, itcauses the artery to dilate. This is followed by activating the powersupply 30, thereby creating a current between the electrode 28 and theelectrode 31 which passes through the balloon wall 26. This currentdrives or drags the NO precursor within the chamber 27 across the walland into contact with the surrounding vessel wall 15 and vasculartissue.

In FIG. 10 is shown a device 30 comprising a catheter 32 carrying a meshstent 34 encircling balloon 36 in its collapsed state. The mesh stent 34is covered with a slow release layer of L-arginine containingpoly(glycolide-lactide) 38. The coronary artery vessel 40 is shown withthe lesion partially closing the coronary vessel artery. In FIG. 11, theballoon 42 has been expanded so as to expand stent 44 to press againstthe vessel wall 46 and open the vessel lumen 48. The coating 50 on thestent 44 can now release the NO precursor agent directly into the vesselwall to inhibit vascular smooth muscle proliferation.

The stent is introduced into the appropriate position as previouslydescribed for directing the balloon for angioplasty. However, in thiscase, the balloon is surrounded by the stent. As indicated above, whenthe balloon and stent are appropriately positioned, the balloon isexpanded expanding the vessel and the stent, and the stent assumes itsexpanded position and is held in place. By using a porous stent, theballoon can also provide the NO precursor agent as previously described.After administration of the NO precursor agent from the balloon, theballoon is deflated and retracted, leaving the stent in position tomaintain the release of the NO precursor agent.

The following examples are offered by illustration, and not by way oflimitation.

EXPERIMENTAL

Methods

Animals

27 male New Zealand white rabbits (NZW) weighing 3.8±1.5 kg, wereentered into the study after one week period of acclimation in thehousing facilities of the Stanford Department of Comparative Medicine.All animals were inspected prior to the study by a veterinarian, andmonitored daily by technicians and investigators. The experimentalprotocols were approved by the Administrative Panel on Laboratory AnimalCare of Stanford University and were performed in accordance with therecommendations of the American Association for the Accreditation ofLaboratory Animal Care.

Animals were then fed a high cholesterol diet (1%, Dyets, Bethlehem,Pa.) for five weeks. Two protocols (acute and chronic study) werecarried out as follows: (FIGS. 1A & B) Acute Study (n=13: protocol seeFIG. 1A). This study was performed to determine if intramuraladministration of the NO precursor L-arginine could enhance local NOsynthesis.

Anesthesia and Surgical Preparation

Six days after initiating the high cholesterol diet, the rabbits wereanesthetized using a mixture of ketamine (5 mg/kg) and rompun (35mg/kg). The right carotid artery was exposed, carefully incised and atygon sheath (5 French in diameter) was inserted under fluoroscopiccontrol into the descending aorta. An angioplasty balloon (ACS, balloondiameter 3 mm) was advanced into either iliac artery and inflated distalto the deep femoral artery at 8 ATM for 6 times with 30 secondincrements between each inflation. Subsequently, the same procedure wasrepeated in the contralateral iliac artery. After four additional weeksof diet, the animals were anesthetized and the left carotid arterycannulated for catheterization and local drug delivery.

Local Drug Delivery

A local drug delivery balloon (3 mm, Dispatch®, Scimed) was advanced tothe left or right iliac artery and placed at the same position as theprevious balloon injury. The proximal end of the delivery catheter wasplaced at the internal iliac branch under fluoroscopic control forlandmark reference. The balloon was inflated to six atmospheres andL-arginine (800 mg/5 ml), or saline was infused for 15 minutes at a rateof 0.2 ml/minute. Subsequently, this procedure was repeated in thecontralateral iliac artery. The iliac artery to receive argininetreatment was randomly determined. An intravenous bolus injection ofKEFZOL® was given for prevention of infections.

Identification of Endothelium-Dependent Vasomotion and QuantitativeAngiography

After local administration of arginine or saline, a control angiogramwas obtained. Subsequently, two infusions containing acetylcholine(10⁻⁵, 10⁻⁶ M) were administered at a rate of 0.8 ml/minute for 3minutes through a Swan Ganz catheter (4 French in diameter), placedabove the iliac bifurcation. Immediately following each infusion, anangiogram of the iliac arteries was performed. All angiograms weremeasured blindly by two investigators with an electronic caliper system.The diameter was measured at three predetermined sites along the area ofdrug delivery at baseline and after each dose of acetylcholine beforeand after the local drug delivery. The vessel diameter was also measuredat a reference site distal to the infusion segment to verify downstreameffects of locally delivered L-arginine. The percent variation indiameter compared to baseline was calculated for each dose and expressedin mean±SEM.

Harvesting of Tissue

30 to 60 minutes following the local delivery of L-arginine, animalswere sacrificed and the iliac arteries carefully freed from adjacenttissue. Care was taken to harvest the exact portion of the artery wherethe local delivery was carried out by matching the anatomy with therespective fluoroscopic picture. To verify the amount of cell damageinduced by the local drug delivery balloon, electron microscopy of thedelivered segment was performed in three rabbits.

Measurements of Nitrogen Oxide

The harvested iliac artery rings were placed in cold physiologicalsolution. The vessel was opened longitudinally and incubated in 2 ml ofHanks buffered saline (HBSS) medium (Irvine Scientific) containingcalcium ionophore (1 μmol/L, A23187, Sigma, St. Louis, Mo.) andL-arginine (100 μl/L, Sigma, St. Louis, Mo.) at 37° C.

At selected time points (0, 30, 60, 120 minutes), samples of the mediumwere collected for measurements of nitrogen oxide (NOx) and replacedwith 2 ml of fresh media. After incubation, the segment was weighed andNOx was measured with a commercially available chemiluminescenceapparatus (model 2108, Dasibi). 100 μl of the samples were injected intoa reduction chamber containing boiling acidic vanadium (III). In thereduction chamber, NO₂ - and NO₃ - are reduced to NO, which is thenquantified by the chemiluminescence detector after reaction with ozone.Signals from the detector were analyzed by a computerized integrator andrecorded as areas under the curve. Standard curves for NO₂ /NO₃ werelinear over the range of 50 pmol to 10 nmol.

Chronic Study (n=14, protocol see FIG. 1B)

This study was performed to determine if a single intramuraladministration of L-arginine could induce a persistent augmentation ofNO activity and inhibit myointimal hyperphasia and/or macrophageaccumulation. One week after initiation of the diet, a balloon injury ofthe iliac arteries was performed under anesthesia. Immediatelythereafter, L-arginine was administered into the wall of the right orthe left iliac artery by the local delivery system. Saline wasadministered using the same catheter system to the contralateral iliacartery. The dose of L-arginine and the infusion rate was identical tothat used in the acute study. One, two, or four weeks (n=4, 4, and 6respectively) after balloon injury and local drug delivery,endothelium-dependent vasomotion was assessed angiographically and/orvessels were harvested for histomorphometric measurementsimmunohistochemistry, or chemiluminescence.

Morphometric Analysis (Intima/Media Ratio)

The harvested vessels were fixed in 10% buffered formalin and thenembedded in paraffin. The embedded vessels were sectioned into thinslices and stained with hematoxylin and eosin for light microscopy andhistomorphometry. Measurements of intimal and medial cross-sectionalarea were made by experienced observers blinded to the treatment group.Histologic cross-sections were scanned with one magnification anddigitized, using the Image Analysist. The following borders werehighlighted with a trackball: external elastic lamina, internal elasticlamina, lumen/intima border. Cross sectional areas of the respectivevessel wall layers were then calculated and an intima/media ratiocalculated. The media was defined as the area between the external andthe internal elastic lamina, the intima was defined as the vessel layerbetween the internal elastic lamina and the intimal/luminal border.

Immunohistochemistry

Immunohistochemical analysis was performed on tissue fixed informaldehyde and embedded in paraffin as described above. Antibodiesagainst rabbit macrophage (RAM 11, Dako Corp., Carpenteria, Calif.) wasused to identify macrophages. Sections were incubated with the primaryantibody for one hour at room temperature, anti-rabbit IgG (biotinconjugate) for 30 minutes and avidin peroxidase for 20 minutes.Peroxidase was then visualized with chromogen (Zymed Laboratories, Inc.,South San Francisco, Calif.). Three respective cross-sections wereimmunostained for each vessel segment treated with either L-arginine orsaline. Macrophage staining was assessed by two experienced observersusing a light microscope. Areas of the vessel defined as media andintima and the percent of the vessel stained for macrophage wasdetermined.

Nitrogen/Oxide Measurements

In four rabbits, tissue was harvested one week following the localdelivery of L-arginine for measurements of NOx levels. Chemiluminescencemeasurements were made as described above.

Data Analysis

Data are expressed as mean±SEM. The difference in vasoreactivity toacetylcholine was expressed as percentage variation in diameter comparedto baseline. The mean change of all arteries in each treatment group(L-arginine or saline) was used for comparison. An unpaired t-test wasperformed to compare values between the two-treatment groups for eachdose of acetylcholine before and following either L-arginine or saline.Additionally, a two-factor analysis of variance was performed to verifythe difference within the treatment group and between the groups.Significantly different changes were assumed at a p-value of ≦0.05.Differences between NOx levels were also identified using Student'st-test with Bonferroni correction for multiple comparisons.

Results

Acute Study (FIG. 1A)

Vasoreactivity

FIG. 2A shows the response of vessel segments to acetylcholine beforeand after the local delivery of L-arginine or saline. Baseline vesseldiameters were identical before and after local drug delivery in bothiliac arteries. There was little change in vessel diameter before localdrug delivery. This probably reflects the fact thatacetylcholine-induced endothelium-dependent vasodilation is attenuatedin the endothelium that regenerate after vascular injury, particularlyin the setting of hypercholesterolemia. After local delivery ofL-arginine, endothelium-dependent vasodilation was restored. Bycontrast, after local delivery of the vehicle, no vasodilation wasobserved. The effect of L-arginine was localized to the segment whichwas exposed to intramural delivery. Almost no change in vessel diameterwas seen at the site distal to the local delivery of L-arginine (FIG.2B). Thus, using this method only local (rather than systemic) effectsof L-arginine were observed.

Nitrogen Oxide Levels

NOx measurements were made in vessel segments harvested 30-60 minutesafter local drug delivery. Vessel segments treated with arginineexhibited a significant increase in nitrogen oxide levels throughout theincubation periods of 30, 60 and 120 minutes.

Chronic Study (FIG. 1B)

Vasoreactivity

Vasomotion studies were performed two or four weeks after the local drugdelivery. Iliac arteries treated with vehicle tended to vasoconstrict inresponse to acetylcholine whereas those treated with L-arginine tendedto vasodilate although the observed differences did not reachstatistical signance (Ach 10⁻¹ M; 2.2±1.3% vs -4.2±3.8%, L-arginine vs.vehicle. Ach 10⁻⁵ ; 7.2±1.0 vs. -4.0±7.5).

Intima Media Ratio

FIG. 5 shows the results obtained two and four weeks following localdrug delivery. Administration of L-arginine significantly inhibitedintimal lesion formation in comparison to vehicle control. Thisphenomenon was even more apparent four seeks following local drugdelivery.

Immunohistochemistry

FIG. 7 shows the percentage of the intimal lesion surface area whichstained positively for macrophages. Only 0-10% of the intimal area wasinfiltrated by positively stained cells in the L-arginine treatedsegments, whereas in vessel segments treated with vehicle, the intimalarea involved by macrophages was markedly higher, in some segmentsexceeding 50% of the intimal area.

Nitrogen Oxide Levels

In vascular segments from four rabbits, NO was measured. NO productionex vivo was significantly higher one week following the delivery ofL-arginine compared to segments exposed to vehicle. These levels werealso higher compared to those achieved one hour following the delivery(FIG. 4).

It is obvious from the above results, that the subject methodology anddevices provides an alternative treatment to substantially reduce theoccurrence of restenosis after vascular injury. The methodology issimple, can be performed in conjunction with the procedure resulting inthe vascular injury, and is found to be effective. Importantly, thisapproach avoids systemic side effects associated with oral orintravenous administration of L-arginine, while providing effectivetreatment. In this way, a procedure which has been commonly used canfind expanded application as a result of the reduced incidence ofrestenosis.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method for reducing the probability ofrestenosis resulting from vascular injury, said methodcomprising:introducing intramurally proximal to the site of said injuryat a time from the time of said injury to a time not later than 6 hoursthereafter, in an aqueous medium a nitric oxide percursor agentconsisting of at least one of L-arginine or L-lysine in an amount toincrease the amount of nitric oxide formation of the cells proximal tosaid injury, whereby a reduced incidence of restenosis occurs.
 2. Amethod for reducing the probability of restenosis resulting fromvascular injury, said method comprising:introducing intramurallyproximal to the site of said injury over a period of about 2 min to 0.5h a nitric oxide percursor agent consisting of at least one ofL-arginine or L-lysine in an amount to increase the amount of nitricoxide formation of the cells proximal to said injury, whereby a reducedincidence of restenosis occurs.
 3. A method for reducing the probabilityof restenosis resulting from vascular injury, said methodcomprising:introducing L-arginine intramurally proximal to the site ofsaid injury at a time from the time of said injury to a time not laterthan 6 hours thereafter as an aqueous solution at a concentration in therange of 20 to 500 g/l to provide an amount to increase the amount ofnitric oxide formation of the cells at the site of said injury, wherebya reduced incidence of restenosis occurs.
 4. A method according to claim3, wherein said introducing is by means of a local delivery catheter. 5.A method for reducing the probability of restenosis resulting frominjury caused by angioplasty or atherectomy, said methodcomprising:introducing intramurally at the site of said injury a stent,which has a body which comprises L-arginine, at the time of said injuryto provide an amount to increase the amount of nitric oxide formation ofthe cells at the site of said injury.
 6. A stent having a bodycomprising a NO precursor agent consisting of at least one of L-arginineor L-lysine releasable under conditions present in a blood vessel.
 7. Amethod for reducing the probability of restenosis resulting fromvascular injury, said method comprising:introducing intramurallyproximal to the site of said injury at the time of said injury, a nitricoxide precursor agent consisting of at least one of L-arginine orL-lysine in an amount to increase the amount of nitric oxide formationof the cells proximal to said injury, without further introduction ofsaid nitric oxide precursor agent, whereby a reduced incidence ofrestenosis occurs.
 8. A method for reducing the probability ofrestenosis resulting from vascular injury, said methodcomprising:introducing intramurally proximal to the site of said injuryby means of a local delivery catheter at a time from the time of saidinjury to a time not later than 6 hours thereafter, a nitric oxidepercursor agent consisting of at least one of L-arginine or L-lysine inan amount to increase the amount of nitric oxide formation of the cellsproximal to said injury, whereby a reduced incidence of restenosisoccurs.